Summary: The current stocks of VIG are limited and outdated. Scientists at CBER and elsewhere (MPH) were encouraged to produce new lots of VIG and subject them to careful titration in validated neutralization assays. In addition, experiments are underway to compare the neutralization capacity of different VIG subclasses, in order to optimize viral neutralization in future production of VIG.The current neutralization assay for vaccinia (plaque-reduction), is labor intensive, slow (up to 7 days), and depends on scoring plaques by eye (i.e., subjective). This assay is difficult to validate and to transfer to sponsor-based laboratories. As an alternative approach, our laboratory initiated late last year the development of an alternative vaccinia neutralization assay, which is fast, quantitative, and easy to perform and transfer to other laboratories.This assay is based on the use of recombinant vaccinia virus expressing a bacterial gene coding for the enzyme b-Gal under the control of the vaccinia late promoter. Several recombinant viruses were tested and two of them, vSC8 and vSC56 are currently under investigation in our laboratory. We also tested several cell substrates for the assay: BSC1, Vero, and HeLa. Results: - The vSC56 recombinant vaccinia virus,expressing the beta-Gal gene under a synthetic promotor, was chosen for our assays. It is used at a 0.05 pfu/Hela cell . All viral neutralization assays are conducted for 18 hr in the absence or presence of multiple dilutions of VIG commercial lots or IVIG, RSVIG lots. -Thus far, it was found that the MPH VIG is somewhat better than the Baxter (4oC) VIG with ID(50) values of 10-15 ug/ml and 25 ug/ml, respectively. -We also tested Ig subclasses derived from the MPH VIG and found that the neutralization efficiency of the IgG1 subclass is superior to all other subclasses and to the total (unseparated) VIG preparation. -We started to test multiple lots of IVIG from 5 different manufacturers. Several lots were found to have significant anti-vaccinia neutralization titers.